Neuroscience 144 (2007) 263–274 MORPHINE PRIMING IN RATS WITH CHRONIC INFLAMMATION REVEALS A DICHOTOMY BETWEEN ANTIHYPERALGESIC AND ANTINOCICEPTIVE PROPERTIES OF DELTORPHIN L. GENDRON,a1 M. J. ESDAILE,a F. MENNICKEN,b strongly reduced the antinociceptive efficacy of the drug H. PAN,d D. O’DONNELL,b J.-P. VINCENT,e L. A. DEVI,d (tail-flick test; % maximum possible antinociceptive effect versus 66.6%؎6.3% in rats injected with %3.6؎%29.6؍(C. M. CAHILL,c T. STROHa AND A. BEAUDETa* (MPE a CFA alone) suggesting that the latter, but not the former, is Department of Neurology and Neurosurgery, Montreal Neurological ␦ Institute, Room 896, 3801 University Street, McGill University, Mon- linked to the OR trafficking events observed neuroanatomi- treal, Quebec, Canada H3A 2B4 cally. These results demonstrate that in chronic inflamma- tion, the antihyperalgesic effects of ␦OR agonists may be b AstraZeneca R&D Montreal, Ville St.-Laurent, Quebec, Canada H4S enhanced by morphine pre-treatment. They also reveal a di- 1Z9 chotomy between mechanisms underlying antihyperalgesic cDepartment of Pharmacology and Toxicology, Botterell Hall, Queen’s and antinociceptive effects of ␦OR agonists. © 2006 IBRO. University, Kingston, Ontario, Canada K7L 3N6 Published by Elsevier Ltd. All rights reserved. dDepartment of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, NY 10029, USA Key words: delta opioid receptor, deltorphin, chronic inflam- mation, antihyperalgesia, antinociception, tolerance. eInstitut de Pharmacologie Moléculaire et Cellulaire, Unité Mixte de Recherche 6097 du CNRS, 06560 Valbonne, France Agonists acting through the mu opioid receptor (␮OR), Abstract—We previously showed that prolonged morphine such as morphine and its derivatives, induce potent anal- treatment and chronic inflammation both enhanced delta gesic effects (Bodnar and Klein, 2004). However, they also opioid receptor (␦OR) cell surface density in lumbar spinal give rise to undesirable side-effects such as nausea, con- cord neurons. Here, we sought to determine whether ad- stipation, and respiratory depression (Colpaert, 1996; ministration of morphine to rats with chronic inflammation Kreek, 1996). In addition, chronic stimulation of ␮OR in- would further increase the bio-availability of ␦OR, and ␦ duces tolerance and physical dependence (Cowan et al., thereby the analgesic properties of the OR agonist deltor- ␦ phin, over that produced by inflammation alone. We found 1988). By contrast, drugs acting on OR produce more that chronic inflammation produced by injection of com- limited analgesia, but also give rise to considerably less plete Freund’s adjuvant (CFA) into the hind paw resulted in undesirable side-effects and induce virtually no tolerance a bilateral increase in the binding and internalization of (Porreca et al., 1984; May et al., 1989; Sheldon et al., fluorescent deltorphin in neurons of the lumbar spinal cord 1990; Szeto et al., 1999; Gallantine and Meert, 2005). For as did prolonged morphine treatment [Morinville A, Cahill these reasons, ␦OR agonists have been proposed as pos- CM, Aibak H, Rymar VV, Pradhan A, Hoffert C, Mennicken F, sible alternatives to ␮OR agonists for the treatment of Stroh T, Sadikot AF, O’Donnell D, Clarke PB, Collier B, Henry JL, Vincent JP, Beaudet A (2004a) Morphine-induced chronic pain, including neuropathic (Mika et al., 2001; Petrillo changes in delta opioid receptor trafficking are linked to et al., 2003; Morinville et al., 2004a) and chronic inflammatory somatosensory processing in the rat spinal cord. J Neuro- pain (Desmeules et al., 1993; Stewart and Hammond, 1994; sci 24:5549–5559]. This effect was accompanied by an Fraser et al., 2000; Hurley and Hammond, 2000; Qiu et al., increase in the antinociceptive efficacy of intrathecal del- 2000; Cahill et al., 2003; Petrillo et al., 2003). torphin as measured using the tail-flick test. Treatment of One of the reasons for the relatively poor analgesic CFA-injected rats with morphine decreased the cell surface ␦ ␦ efficiency of OR agonists may be that only a small pro- availability of OR in neurons of the dorsal horn of the ␦ lumbar spinal cord as compared with treatment with CFA portion of OR is actually present on neuronal plasma alone. Behaviorally, it significantly enhanced the antihy- membranes under baseline conditions (Cheng et al., 1995, peralgesic effects of deltorphin (plantar test; % maximum 1997; Elde et al., 1995; Zhang et al., 1998; Cahill et al., -2001a). However, under conditions of chronic inflamma %32.4؎%113.5؍(possible antihyperalgesic effect (MPAHE versus 26.1%؎11.6% in rats injected with CFA alone) but tion, such as produced in rodents by injection of complete 1 Present address: Department of Physiology and Biophysics, Faculty Freund’s adjuvant (CFA) in the hind paw, we observed a of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada, massive recruitment of ␦OR from intracellular stores to the J1H 5N4. plasma membrane in neurons of the dorsal horn of the ϩ ϩ *Corresponding author. Tel: 1-514-398-1913; fax: 1-514-398-5871. spinal cord (Cahill et al., 2003; Morinville et al., 2004b). E-mail address: [email protected] (A. Beaudet). Abbreviations: CFA, complete Freund’s adjuvant; DRG, dorsal root This increase in the pharmacological availability of ␦OR ganglion; Fluo-DLT, ␻-Bodipy 576/589 deltorphin-I 5-aminopentylam- was postulated to account for the enhanced antihyperal- ide; i.t., intrathecal/intrathecally; MPAHE, maximum possible antihy- gesic efficacy of the centrally administered ␦OR agonists peralgesic effect; MPE, maximum possible antinociceptive effect; MS, morphine sulfate; ␮OR, mu opioid receptor; PB, phosphate buffer; reported in conditions of chronic inflammation (Hylden RIA, radioimmunoassay. et al., 1991; Stewart and Hammond, 1994; Fraser et al., 0306-4522/07$30.00ϩ0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.08.077 263 264 L. Gendron et al. / Neuroscience 144 (2007) 263–274 2000; Hurley and Hammond, 2000; Cahill et al., 2003; were carried out 12 h after the last morphine or saline injection. To Morinville et al., 2004b). The CFA-induced up-regulation of determine the dose-response of morphine pre-treatment on the cell surface ␦OR in spinal cord neurons involves the par- antinociceptive efficacy of deltorphin, additional groups of naïve rats were treated: (1) for 48 h with half (1, 3, 5, and 8 mg/kg; low ticipation of ␮OR as it can no longer be elicited in ␮OR dose) or twice (10, 15, 20 and 30 mg/kg; high dose) the standard knockout mice (Morinville et al., 2004b). The effect of morphine dose; or (2) for 96 h with a modified standard morphine chronic inflammation on ␦OR trafficking is therefore akin to dose (5, 8, 10, 10, 10, 10, 10, 15 mg/kg). that of prolonged treatment with ␮OR-preferring agonists (including morphine, fentanyl, methadone and etorphine), In vivo ␦OR internalization assay which was shown to induce a selective membrane recruit- ␦ ␦ To assess the cell surface availability of OR in neurons of the ment of OR in neurons of layers I–VI of the rat lumbar lumbar spinal cord (L4-5), naïve rats (nϭ3), rats treated with CFA spinal cord (Cahill et al., 2001b; Morinville et al., 2003). As 72 h earlier (nϭ3), and rats treated with CFA and subjected to in animals with chronic inflammation, this increased den- prolonged treatment with morphine (standard dose; nϭ3) were sity of cell surface ␦OR translates into a potentiation of the injected i.t. with 30 ␮lof␻-Bodipy 576/589 deltorphin-I 5-amin- antinociceptive properties of deltorphin II injected intrathe- opentylamide (Fluo-DLT) as described (Morinville et al., 2004a). cally (i.t.) (Cahill et al., 2001b; Morinville et al., 2003). Briefly, animals were anesthetized with sodium pentobarbital (Somnotol; MTC Pharmaceuticals, Cambridge, Ontario, Canada, However, it is unclear whether chronic inflammation and 6.5 mg/100 g of body weight) and injected i.t. via a lumbar punc- sustained morphine treatment affect the same neuronal ture with 0.8 nmol Fluo-DLT diluted in 30 ␮l saline at the L5–L6 populations in the lumbar spinal cord and whether their intervertebral space. Appropriate placement of the needle was effects are independent or cumulative. validated by the observation of a little flick of the tail. Twenty The aim of the present study was therefore to investi- minutes after injection of the fluorescent ligand, rats were killed by gate the distribution of neurons which up-regulate cell intra-aortic arch perfusion of, in succession, 4% paraformalde- hyde (PFA) in 500 ml 0.1 M phosphate buffer (PB, pH 7.4) at 4 °C surface ␦OR in the lumbar spinal cord in response to and 100 ml each of 10%, 20%, and 30% sucrose in 0.2 M PB (pH chronic inflammation of the hind paw and to determine 7.4). The lumbar segment of the spinal cord was snap-frozen in whether this up-regulation is affected by a morphine treat- isopentane at Ϫ45 °C and stored at Ϫ80 °C until sectioning. ment in an attempt to determine if pre-treatment of animals Tissues were sectioned on a cryostat at a thickness of 20 ␮m and with morphine (to heighten the cell surface density of ␦OR) thaw-mounted onto chrome alum/gelatin-coated slides (without would further enhance the analgesic efficacy of ␦OR ago- coverslips). nists in the treatment of chronic pain. Neurons having specifically bound and internalized Fluo-DLT (characterized by the presence of intra-cytoplasmic fluorescent puncta) were visualized using a Zeiss confocal laser scanning EXPERIMENTAL PROCEDURES microscope LSM510 (Carl Zeiss Canada Ltd., Toronto, Ontario, Canada) equipped with an inverted microscope (oil-immersion Animals objectives, 25ϫ,40ϫ and 63ϫ) and a He/Ne laser with an exci- tation wavelength of 543 nm. For each animal, five representative All experiments were carried out in adult male Sprague–Dawley images were acquired on each side of the lumbar spinal cord, rats (220–280 g; Charles River, St-Constant, Quebec, Canada), contralateral and ipsilateral to the inflamed paw, in each of lami- maintained on a 12-h light/dark cycle.